Method of manufacturing compound with biocatalyst by using controlled reaction temperature

ABSTRACT

Provided is a method for producing a compound using a biocatalyst. In the method for continuously producing a compound using a biocatalyst in one or a plurality of reaction tanks, the downstream reaction temperature is set higher than the upstream reaction temperature of the flow of the catalyst in a reaction tank or between reaction tanks.

TECHNICAL FIELD

The present invention relates to a method for producing a compound usinga biocatalyst.

BACKGROUND TECHNIQUE

Biocatalysts, such as cells, immobilized cells, or immobilized enzymes(these may be hereinafter referred to as “biocatalysts”) have advantagesin that, for example, reaction processes can be simplified, the reactionproducts have high purity due to a reduced amount of by-productsgenerated, and highly-reactive materials can also be stably produced dueto a mild reaction condition. Thus, biocatalysts are recently used inthe production of many compounds.

Biocatalysts, however, cause lowering (deactivation) in the catalyticactivity during a reaction. Therefore, methods for regulating thedeactivation have been studied in order to enhance the amount of thecompound produced per unit amount of catalyst, that is, the productivityof the catalyst (hereinafter, simply referred to as “productivity”). Forexample, those methods include: a method in which a reaction is carriedout at low temperature from the freezing point to 15° C. (JapanesePatent Examined Publication (kokoku) No. 56-38118); a method in whichlow-concentrated substrates are continuously supplied through aplurality of feed hoppers (Japanese Patent Examined Publication (kokoku)No. 57-1234); a method in which microorganisms or a treated productthereof are processed with an organic solvent (Japanese PatentApplication Laying-Open (kokai) No. 5-308980); a method in which areaction is carried out in the presence of higher unsaturated fattyacids (Japanese Patent Application Laying-Open (kokai) No. 7-265090);and a method in which cells are cross-linked with glutaraldehyde or thelike (Japanese Patent Application Laying-Open (kokai) Nos. 7-265091 and8-154691).

DISCLOSURE OF THE INVENTION

These methods by themselves, however, do not sufficiently enhance theproductivity of a catalyst and, as a result, the amount of the catalystused in the production of a compound is not negligible. This results innot only the increased production cost of a compound but also theincreased amount of disposable catalysts and, thus, a method fordisposing thereof becomes a concern.

Accordingly, the object of the present invention is to provide a methodfor producing a compound in a cost-effective, efficient, andenvironmentally-friendly manner, which more effectively utilizes abiocatalyst by improving a productivity of a catalyst, thereby loweringa proportional cost of a catalyst in the production of a compound andgenerating less waste.

In general, a reaction is preferably carried out at lower temperature inorder to prevent the deactivation when producing a compound using abiocatalyst. However, the present inventors have conducted concentratedstudies and, as a result, found that the productivity of the catalystwas improved by raising a downstream temperature than the upstreamtemperature of the flow of the catalyst in a reaction tank. This has ledto the completion of the present invention. Further, they have foundthat the present invention could be attained by a technically andeconomically simple manner, that is, a decrease in the amount of heatremoved from the reaction tank is sufficient when the reaction is anexothermic reaction.

More specifically, the present invention is as follows:

(1) A method for continuously producing a compound using a biocatalystin one or a plurality of reaction tanks, wherein a downstream reactiontemperature is set higher than a upstream reaction temperature in areaction tank or between reaction tanks;

(2) The production method according to (1), wherein the downstreamreaction temperature is set higher than the upstream reactiontemperature by at least 1° C. in a reaction tank or between reactiontanks;

(3) The production method according to (1), wherein the downstreamreaction temperature is set higher than the upstream reactiontemperature by at least 5° C. in a reaction tank or between reactiontanks;

(4) The method for producing a compound according to any one of (1) to(3), wherein the biocatalyst is microbial cells or a processed productthereof;

(5) The method for producing a compound according to (4), wherein thecompound to be produced is an amide compound;

(6) The method for producing a compound according to (5), wherein thecompound to be produced is acrylamide, nicotinamide, or5-cyanovaleramide; and

(7) The method for producing a compound according to any one of (1) to(6), wherein the catalyst flows in parallel with the flow of thereaction solution in the reaction tank.

The present invention will be described in more detail below.

The present invention is applied to a method for continuously producinga compound in a reaction tank using a biocatalyst. A method forcontinuously producing a compound in a reaction tank using a biocatalystrefers to a method for producing a compound using a bioreactor includinga biochemical reactor (an enzyme reactor) and a biological reactor (amicrobiological reactor) and can be carried out using various types ofreactors such as a reactor of stirred tank type, fixed bed type,fluidized bed type, or moving bed type. In this method, the reaction forproducing a compound is carried out in a reaction tank. Some reactionsutilize only one reaction tank and some other reactions utilize aplurality of reaction tanks. Preferably, two or more reaction tanks areutilized from the viewpoints of, for example, the improvement in theoperability such as temperature control and an easiness of catalystsubstitution and the improvement in the reaction efficiency.

The biocatalysts used in the present invention include animal cells,plant cells, organelles, cells (viable cells or killed cells) containingan enzyme which catalyzes the reaction of interest, or a processedproduct thereof. The processed product includes a crude or refinedenzyme extracted from cells, and an immobilized product of animal cells,plant cells, organelles, cells (viable cells or killed cells), orenzymes per se by, for example, an entrapping method, a cross-linkingmethod, or a carrier binding method. Cells used as biocatalysts includemicrobial cells such as Rhodococcus rhodochrous and Pseudomonaschlororaphis and, as the enzyme, nitrile-hydratase produced from thesemicroorganisms is included. The “entrapping method” used herein is amethod in which cells or enzymes are enveloped into fine lattices ofpolymer gels or covered with a semipermeable polymeric coat. The“cross-linking method” is a method in which an enzyme is cross-linkedwith a reagent having two or more functional groups (a polyfunctionalcross-linking agent). The “carrier binding method” is a method in whichan enzyme is bound to a water-insoluble carrier. Immobilizing carriersused in the immobilization include glass beads, silica gel,polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginicacid, agar, and gelatin.

The entrapping-immobilization method is widely applied to the industrialuse among methods for immobilizing cells because it can provideimmobilized cells having high cell concentration. For example, JapanesePatent Examined Publication (kokoku) No. 58-35078 and Japanese PatentApplication Laying-Open (kokai ) No. 7-203964 disclose an example inwhich an acrylamide and/or an acrylamide derivative are used as amonomer for entrapping-immobilization. The compound produced accordingto the present invention is not particularly limited as long as thecompound can be produced by a biocatalytic action. Examples of compoundsinclude general-purpose chemicals such as alcohols and amides, andfoods, perfumeries, and medicines such as amino acid, antibiotics, andphysiological substances or a raw material or intermediate productthereof. In particular, in the production of general-purpose chemicals,it is important and indispensable to decrease the amount of catalystsused from the economical point of view. Thus, the present invention ispreferred in the production of general-purpose chemicals usingbiocatalysts and, more specifically, is more preferred in the productionof amide compounds, which are general-purpose chemicals recentlymass-produced using biocatalysts. Examples of amide compounds includeacrylamide, nicotinamide, and 5-cyanovaleramide.

The method for continuously producing a compound according to thepresent invention is a method in which a starting compound iscontinuously or intermittently added to a reaction tank whilecontinuously or intermittently removing a reaction solution therefromwithout removing the total amount of the reaction solution from thereaction tank. Specifically, it does not refer to the production inwhich the total amount of the reaction solution is periodically removed,i.e., the production that is called a batch reaction or semibatchreaction. Possible reaction modes for continuously producing a compoundusing a biocatalyst according to the present invention include a modewhich utilizes a fixed-bed, moving-bed, fluidized-bed, stirred tank andthe like. In any of these modes, the reaction tank used in the presentinvention is preferably equipped with a cooling or heating system suchas a jacket, a cooling or heating coil, an external cyclic coolingsystem or external cyclic heating system. Alternatively, the reactor asa whole or a part thereof may be immersed in a constant-temperature bathto realize cooling or heating. A heat exchanger can also be insertedbetween reaction tanks.

In such a mode, a biocatalyst is deactivated over the elapse of the timein any case. Thus, the biocatalyst should be continuously orintermittently added to a reaction tank while being removed from thereaction tank. Therefore, in the continuous production method, a givenflow of the biocatalyst from an upstream toward a downstream isgenerated. The “upstream” according to the present invention refers to aside to which catalysts are added to a reaction tank and, conversely,the “downstream” refers to a side from which the catalysts are removed.

Each reaction mode will be described in more detail. In the case of afixed bed, it is supposed that a fixed bed comprising a plurality oftanks should be used as a pseudo-moving bed (used in a merry-go-roundsystem), and in the case of a fluidized bed or a stirred tank, it shouldbe a series of plural tanks. In this case, the upstream reaction tank isa reaction tank which is located on the side to which catalysts areadded among a plurality of reaction tanks, and the downstream reactiontank is a reaction tank which is located on the side from whichcatalysts in the reaction system are removed. In the case of a movingbed in which a biocatalyst moves along with the flow of a reactionsolution in one reaction tank, the “upstream” refers to a vicinity ofwhat is called an inlet of a reaction tank to which catalysts are added,and the “downstream” refers to a vicinity of what is called an outlet ofa reaction tank from which catalysts in the reaction system are removed.The “moving bed” used herein includes the reaction mode utilizing a flowtubular reaction tank.

Accordingly, “the downstream reaction temperature is set higher than theupstream reaction temperature of the flow of the catalyst in thereaction tank” means that the downstream reaction temperature is sethigher than the upstream reaction temperature of the above-describedcatalyst. More specifically, in a serial mode utilizing a plurality oftanks, the temperature of the reaction tank located at the lowermoststream among a plurality of tanks is set higher than the temperature ofthe reaction tank located at the uppermost stream. For example, whenfour reaction tanks are connected, the temperature of the fourthreaction tank is set higher than that of the first tank. In a serialmode utilizing a single tank such as a moving bed, the temperature ofthe portion near the outlet of the reaction tank is set higher than thetemperature of the portion near the inlet of the reaction tank. Thephrase “the reaction temperature is higher” used herein means that thetemperature can be confirmed to be higher in a measurable range, i.e.,higher by at least 0.1° C. In order to make the present invention moreeffective, the temperature is preferably higher by at least 1° C., andis more preferably higher by at least 5° C. The reaction temperature isadequately selected taking the stability of the catalyst used in thereaction and the like into consideration.

In the present invention, the flow of a catalyst is preferably inparallel with the flow of a reaction solution. The flow of a catalyst isin parallel with the flow of a reaction solution means that the flow ofthe reaction solution is toward the same direction as the flow of thecatalyst. When the reaction is an exothermic reaction if the catalystflows in the same direction as the reaction solution, any regulation ofthe amount of heat removed is sufficient for raising the reactiontemperature and, thus, the downstream reaction temperature can be easilyset higher than the upstream reaction temperature of the flow of thecatalyst in the reaction tank.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail with reference tothe following examples. These examples, however, are not intended tolimit the technical scope of the present invention.

EXAMPLE 1

(1) Preparation of Biocatalyst

Rhodococcus rhodochrous J1 strain having an activity ofnitrile-hydratase (deposited with the International Patent OrganismDepositary of the National Institute of Advanced Industrial Science andTechnology as of Sep. 18, 1987 under the accession number FERM BP-1478(Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan)) wasaerobically cultured in a medium (pH 7.0) containing 2% of glucose, 1%of urea, 0.5% of peptone, 0.3% of yeast extract, and 0.05% of cobaltchloride (all values are by mass) at 30° C. The cultured product washarvested using a centrifuge and washed with 50 mM phosphate buffer (pH7.0) to obtain a cell suspension (15% by mass of dry cell).

(2) Reaction from 3-cyanopyridine to Nicotinamide

Four jacketed separable flasks (interior volume of 1 L) were connectedin series. To the first tank were continuously added 50 mM phosphatebuffer (pH 8) having 15% 3-cyanopyridine dissolved therein at a flowrate of 200 ml/hr and a cell suspension at a flow rate of 0.3 ml/hr and,while stirring, a reaction was carried out while regulating the reactiontemperature using coolant (20° C.) of the jacket so as to bring thetemperatures of the first to the fourth reaction tanks to 30° C., 30°C., 32° C., and 35° C., respectively.

Three days later, the reaction solution discharged from the fourth tankwas assayed by liquid chromatography (column: ODS-80A (GL Sciences Inc.,eluant: a 5% acetonitrile/10 mM phosphate buffer (pH 7), detection: 200nm)). As a result, 3-cyanopyridine was not detected while about 17%nicotinamide was detected.

Comparative Example 1

A reaction was carried out using the cell suspension prepared in Example1 in the same manner as used in Example 1, except that the reactiontemperature was set at 30° C. for all of four tanks.

Three days later, the reaction solution discharged from the fourth tankwas assayed in the same manner by liquid chromatography. As a result,only 16% of nicotinamide was generated and about 1% of unreacted3-cyanopyridine was detected.

Comparative Example 2

A reaction was carried out using the cell suspension prepared in Example1 in the same manner as used in Example 1, except that the reactiontemperature was set at 35° C. for all of four tanks.

Three days later, the reaction solution discharged from the fourth tankwas assayed in the same manner by liquid chromatography. As a result,only 15% of nicotinamide was generated and about 2% of unreacted3-cyanopyridine was detected.

EXAMPLE 2

(1) Preparation of Biocatalyst

Pseudomonas chlororaphis B23 having an activity of nitrile-hydratase(deposited with the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology as ofNov. 16, 1981 under the accession number FERM BP-187 (Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan)) was aerobically cultured in amedium (pH 7.5) containing 1.0% of sucrose, 0.5% of methacrylonitrile,0.3% of peptone, 0.1% potassium dihydrogenphosphate, 0.1% dipotassiumhydrogenphosphate, 0.1% of magnesium sulfate, 0.3% of yeast extract, and0.001% of ferrous sulfate (all values are by mass) at 25° C. Thecultured product was washed with 50 mM phosphate buffer (pH 7.0) toobtain a cell suspension (12% by mass of dry cell).

Separately, an aqueous solution of monomer mixture was prepared so as tobring acrylamide, methylenebisacrylamide, and 2-dimethylaminopropylmethacrylamide to 30, 1, and 4% by mass, respectively.

Subsequently, a cell suspension, an aqueous monomer solution, and anaqueous 10% by mass N,N,N′,N′-tetramethyl ethylene diamine solution weresuccessively subjected to line mixing with an aqueous 10% by massammonium persulfate solution at 5, 2, 0.1, and 0.1 L/hr, respectively,for polymerization. Thereafter, the resultant product was cut into about1 mm-square particles to obtain immobilized cell particles. Theseimmobilized cell particles were washed with 50 mM phosphate buffer (pH7.0) by dipping while being fluidized to obtain immobilized cellcatalysts (about 8% by mass of dry cell is contained in this catalyst).

(2) Reaction from Acrylonitrile to Acrylamide Using Immobilized CellCatalyst.

A similar device as used in Example 1 was provided, except that wiregauzes were provided at the outlets of the reaction solution from eachreaction tank to prevent the immobilized cell catalysts from beingdischarged from each reaction tank. 50 g of immobilized cell catalystwas added to each reaction tank. 50 mM phosphate buffer (pH 7) andacrylonitrile were continuously added to the first tank at 155 ml/hr andat 25 g/hr, respectively. Only acrylonitrile was continuously added tothe second tank at 20 g/hr. While stirring in each of these tanks, thereaction temperature was regulated using coolant (5° C.) of the jacketso as to bring the temperature of the first to the fourth reaction tanksto 10° C., 10° C., 12° C., and 15° C., respectively. 6 g of catalyst wasremoved from the fourth tank once a day using wire gauzes forsubstituting catalysts in the reaction tank. 6 g each of catalyst wastransferred from the third tank to the fourth tank, from the second tankto the third tank, and from the first tank to the second tank, and 6 gof catalyst was then added to the first tank, thereby continuouslyperforming the acrylamide-producing reaction.

The reaction solution discharged from the fourth tank was assayed by gaschromatography (column: PraPak-PS (Waters), 1 m, 180° C., carrier gas:nitrogen, detector: FID) once a day. During the operation for aboutthree months, only about 30% of acrylamide was detected while nounreacted acrylonitrile was detected.

Comparative Example 3

A reaction was carried out using the immobilized cell catalyst preparedin Example 2 in the same manner as used in Example 2, except that thereaction temperature was set at 10° C. for all of four tanks.

After about 1.5 months, unreacted acrylonitrile began to remain in thereaction solution discharged from the fourth tank, and the quality ofthe acrylamide product began to deteriorate. The amounts of catalystsadded and removed were changed to 8 g per day, and as a result,unreacted acrylonitrile came to be not detected again.

INDUSTRIAL APPLICABILITY

The present invention can easily decrease the amount of the biocatalystused in the production of a compound and, thus, can provide a method forproducing a compound in a cost-effective, efficient, andenvironmentally-friendly manner, which is capable of lowering theproportional cost of a catalyst in the production of a compound andgenerating less wastes.

All publications cited herein are incorporated herein in their entirety.A person who has ordinary skill in the art would easily understand thatvarious changes and modifications of the present invention are possiblewithout departing from the range of the technical idea and the scope ofthe invention described in the accompanying claims. The presentinvention is also intended to include such changes and modifications.

1. A method for continuously producing an amide compound in the presenceof a biocatalyst in two or more reaction tanks, wherein a downstreamreaction temperature is set higher than an upstream reaction temperatureby at least 1° C. between reaction tanks.
 2. The method according toclaim 1, wherein the downstream reaction temperature is set higher thanthe upstream reaction temperature by at least 5° C. between reactiontanks.
 3. The method according to claim 1, wherein the biocatalyst ismicrobial cells or a processed product thereof.
 4. The method accordingto claim 1, wherein the compound produced is acrylamide, nicotinamide,or 5-cyanovaleramide.
 5. The method according claim 1, wherein thebiocatalyst flows in parallel with a flow of reaction solution in thereaction tanks.
 6. The method according to claim 2, wherein thebiocatalyst is microbial cells or a processed product thereof.
 7. Themethod according to claim 3, wherein the biocatalyst is microbial cells.8. The method according to claim 4, wherein the biocatalyst is microbialcells or a processed product thereof.
 9. The method according to claim5, wherein the biocatalyst is microbial cells or processed productthereof.
 10. The method according claim 2, wherein the biocatalyst flowsin parallel with a flow of reaction solution in the reaction tanks. 11.The method according claim 3, wherein the biocatalyst flows in parallelwith a flow of reaction solution in the reaction tanks.
 12. The methodaccording claim 4, wherein the biocatalyst flows in parallel with a flowof reaction solution in the reaction tanks.
 13. The method accordingclaim 5, wherein the biocatalyst flows in parallel with a flow ofreaction solution in the reaction tanks, and wherein said biocatalyst isa processed product of microbial cells.
 14. The method according claim6, wherein the biocatalyst flows in parallel with a flow of reactionsolution in the reaction tanks.
 15. The method according claim 7,wherein the biocatalyst flows in parallel with a flow of reactionsolution in the reaction tanks.
 16. The method according claim 8,wherein the biocatalyst flows in parallel with a flow of reactionsolution in the reaction tanks.
 17. The method according claim 9,wherein the biocatalyst flows in parallel with a flow of reactionsolution in the reaction tanks.
 18. The method of claim 1, wherein theamide compound is acrylamide.